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Abstract:

The present invention relates to the diagnosis and treatment of cancer,
and specifically to a method of diagnosing the presence or metastasis of
cancer by detecting plasma Hsp90α having the amino acid sequence of
SEQ ID No.1 as a tumor marker. In addition, the present invention also
relates to a method for the treatment of cancer and metastasis.

Claims:

1. An isolated polypeptide comprising or consisting of the amino acid
sequence of SEQ ID No.1.

2. The polypeptide according to claim 1, wherein one or more amino acid
residues in the amino acid sequence of SEQ ID No.1 selected from the
group consisting of Thr90, Ser231, Ser263, Tyr309 and a combination
thereof are phosphorylated.

3. The polypeptide according to claim 2, wherein the Thr90 is
phosphorylated.

4. A method of determining the presence, stage and/or metastasis of a
cancer in a subject comprising the step of detecting the level of a
polypeptide comprising or consisting of the amino acid sequence of SEQ ID
No.1 in a plasma sample of the subject by using an agent specifically
binding to a polypeptide of claim 1.

5. A method of screening the presence of a cancer in a high-risk
population comprising the step of detecting the level of a polypeptide
comprising or consisting of the amino acid sequence of SEQ ID No.1 in a
plasma sample by using an agent capable of specific binding to a
polypeptide of claim 1.

6. A method of determining the prognosis of a patient having a cancer
comprising the step of detecting the level of a polypeptide comprising or
consisting of the amino acid sequence of SEQ ID No.1 in a plasma sample
of the patient by using an agent capable of specific binding to a
polypeptide of claim 1.

7. A method of determining the efficiency of a surgery, radiotherapy or
chemotherapy treatment to a patient having a cancer comprising the step
of detecting the level of a polypeptide comprising or consisting of the
amino acid sequence of SEQ ID No.1 in a plasma sample of the patient by
using an agent capable of specific binding to a polypeptide of claim 1.

9. The method according to claim 4, wherein the agent is a specific
antibody to the polypeptide.

10. The method according to claim 9, wherein the antibody is a monoclonal
antibody or an antigen binding fragment thereof.

11. The method according to claim 10, wherein the antigen-binding
fragment is selected from scFv, Fab, Fab' and F(ab')2.

12. The method according to claim 10, wherein the antibody is MAb E9 or
D10 produced by the cell line deposited under CGMCC No. 2903 or 2904,
respectively.

13. The method according to claim 9, wherein the antibody specifically
binds to the polypeptide present in plasma.

14. The method according to claim 9, wherein the antibody specifically
binds to a phosphorylated form of the polypeptide, said phosphorylated
form of the polypeptide contains one or more phosphorylated amino acid
residues in the amino acid sequence of SEQ ID No.1 selected from the
group consisting of Thr90, Ser231, Ser263, Tyr309 and a combination
thereof.

15. The method according to claim 14, wherein the antibody specifically
binds to the polypeptide which is phosphorylated at Thr90.

16. A method of preventing or treating cancer metastasis in a subject
comprising the step of administering an inhibitor of a polypeptide of
claim 1 to the subject.

17. The method according to claim 16, wherein the inhibitor is a specific
antibody against a polypeptide comprising or consisting of the amino acid
sequence of SEQ ID No.1.

18. The method according to claim 17, wherein the antibody is a humanized
antibody or an antigen binding fragment thereof.

19. The method according to claim 17, wherein the antibody specifically
binds to a phosphorylated form of the polypeptide, said phosphorylated
form of the polypeptide contains one or more phosphorylated amino acid
residues in the amino acid sequence of SEQ ID No.1 selected from the
group consisting of Thr90, Ser231, Ser263, Tyr309 and a combination
thereof.

20. The method according to claim 19, wherein the antibody specifically
binds to the polypeptide which is phosphorylated at Thr90.

21. The method according to claim 16, wherein the antibody is MAb E9 or
D10 produced by the cell line deposited under CGMCC No. 2903 or 2904,
respectively.

Description:

FIELD OF INVENTION

[0001] This invention relates to the diagnosis and therapy of tumor, and
specifically to a novel tumor biomarker and methods and kits for the
detection of cancer occurrence and metastasis. The invention also relates
to methods and medicaments for the treatment of cancer and/or its
metastasis.

BACKGROUND OF INVENTION

[0002] Currently, around 11 million people are diagnosed as tumor patients
every year worldwide, and it is speculated that this number will increase
to more than 16 million by the year of 2020. In 2005, among the 58
million deaths, 7.6 million are caused by cancer (accounting for about
13%). This number is increasing, and it is expected that 9 and 11.4
million people will die of cancer by the year of 2015 and 2030
respectively. (World Health Organization, 2006)

[0003] Tumor markers are the substances produced by tumor cells during the
progression caused by gene mutation, including antigens and other
bio-active substances, which can be used for the early detection of
cancers as well as the monitoring of disease progression and response to
a treatment (ASCO, 1996). It brings huge benefit for the clinical
treatment of cancers, especially when it can be detected before any
obvious clinical phenomenon or when it can be used to monitor the
patients' response to certain treatment. At present, in order to better
meet the clinical need, greater efforts on the research and development
of tumor biomarkers are required.

[0004] The applications of most tumor biomarkers currently used in clinic
are more or less restricted due to the not-so-good sensitivity and
specificity. For example, the AFP level and ultra-sonic examinations are
largely used for liver cancer detection. Although their sensitivities are
not very high, they indeed prolong the survival rate of the patients by
diagnosis of the high-risk people. The tumor antigen CA-125 has a higher
sensitivity but lacks specificity. Similarly, the blood tumor biomarker
CA15-3 which is used for the detection of breast cancer could hardly be
used for early detection due to low sensitivity. Therefore, methods for
the early detection of cancer as well as to distinguish benign and
malignant tumors are currently not available in clinic. New technologies
as well as new methods are required to be developed to resolve these
problems.

[0005] The development of tumor proteonomics brings hope for the
identification of novel tumor biomarkers. The malignant transformation of
tumors always results in the change of protein expressions, which could
be quantified at the protein level. Thus, a lot of information and data
could be derived, by which potential biomarkers could be identified and
evaluated for further development and clinical application.

[0006] Hsp90α (Heat shock protein 90α, Hsp90α) is a
molecular chaperone, which functions to stabilize its client proteins in
their active states. Hsp90α is one of the most abundant proteins in
the eukaryotic cells accounting for about 1-2% of whole cell proteins.
The intracellular Hsp90α mainly functions to stabilize its clients
(i.e. estrogen receptor) and assistant their maturation (i.e. some
kinases and signal proteins). However, in other physiological conditions,
Hsp90α is also involved in mediating events such as the evolution
of mutated proteins, rearrangement of cytoskeleton, translocation of
nuclear proteins, cell proliferation and apoptosis, protein degradation,
antigen processing and LPS recognition etc. Hsp90α is also related
with many diseases such as cancer, autoimmune disorder and cardiovascular
diseases. For example, the monoclonal antibody against the antigen of
LKVIRK sequence derived from Hsp90α can be used to treat
fungal-related infection, and this clinic trial is currently ongoing by
the Neutec company (Trade name: Mycogrip).

[0007] It is also reported that Hsp90α could be secreted under some
stimulus (Liao et al. (2000) J. Biol. Chem. 275, 189-96). As a classical
intracellular protein, there is little report regarding the function of
extracelluar Hsp90α. In previous reports, Hsp90α was
identified to help the antigen processing in APCs and was one of the four
proteins related to the lipid raft. They can interact with LPC thus
trigger the intracellular response of cells. (Triantafilou et al. (2002)
Trends in Immunology 23, 301-4).

[0008] Hsp90α was also found to be highly expressed in the surface
of some tumor cells, including the small-cell-lung cancer cell, melanoma
and liver cancer cells (Ferrarini et al. (1992) Int. J. Cancer 51,
613-19). The high expression of cell surface Hsp90α in these cells
were speculated to be related with the antigen processing while direct
evidence is not available.

[0010] The intracellular Hsp90α is an important target for the
development of anti-cancer drugs, as it is involved in the regulation of
many signaling pathways which are critical for the cancer cell
transformation. Inhibition of intracellular Hsp90α could result in
the selective degradation of proteins related with cell proliferation,
cell cycle control as well as apoptosis. Recently, some known antibiotics
such as Geldanamycin, Radicicol and Coumermycin A1 are natural inhibitors
of Hsp90α. A patent (WO 00/53169) describes this mechanism and
proposes that preventing the interaction of chaperones with its clients
could result in the inhibition of its chaperone activity. Among these
antibiotics, Coumarin and its derivatives are believed to have this
activity. However, these inhibitors described in patent WO 00/53169
mainly target the intracellular Hsp90α.

[0011] The analogue of Geldanamycin 17-AAG is also an inhibitor of
Hsp90α and is currently under clinical trials. However, some
reports show that 17-AAG could have non-specific inhibitory effects and
cell toxicity by interacting with many other cellular components. In
addition, due to the limited knowledge on the physiological functions of
Hsp90α and its clients, direct inhibition of intracellular
Hsp90α is risky.

[0012] The patent (EP1457499A1) describes the function of extracellular
Hsp90α in promoting tumor cell invasion via activating the MMP-2.
Based on these mechanisms, the patent proposes that inhibition of
extracellular Hsp90α could prevent the tumor invasion and
metastasis, and by detecting the response of tumor cells to the treatment
of Hsp90α inhibitor they can deduce the invasive ability of the
cells and their relationship with Hsp90α.

[0013] The inventors of patent WO/2008/070472 propose that they can
monitor the anti-tumor efficacy of Hsp90α targeted therapy by
detecting the plasma Hsp90α and other related factors. In this
patent, they provide the relationship between the plasma Hsp90α
level and the efficacy of the inhibitors including 17-AAG and 17-DMAG as
well as the relationship between the level of plasma Hsp90α and
tumor volume in mouse models. However, they do not provide any evidence
about the exact form of plasma Hsp90α and do not demonstrate the
relationship between the plasma Hsp90α level and tumor malignancy
especially tumor metastasis. They do not propose the application of
plasma Hsp90α as an independent tumor biomarker in tumor diagnosis
and prognosis, either.

[0014] One group reported that serum Hsp90α level is related with
the stages of non-small-cell lung cancer (Xu et al. (2007) J. Cancer Mol.
3, 107-112). The serum level of Hsp90α in these lung cancer
patients was significantly higher than that of normal people or benign
tumor patients. However, again this paper did not identify the exact form
of serum Hsp90α as well as its relationship with tumor metastasis.
Besides, it only investigated non-small-cell lung cancer, while the
relationship between serum Hsp90α level with breast cancer, liver
cancer, and pancreatic cancer is unknown. Moreover, the serum level of
Hsp90α was not quantitatively measured, thus could hardly be
translated to clinic development and further application.

SUMMARY OF INVENTION

[0015] This invention is based on the discovery that the plasma
Hsp90α level is correlated with the development, malignancy and
metastasis of many types of cancer. Accordingly, plasma Hsp90α can
be used as a new tumor biomarker. The inventors found that the plasma
Hsp90α is different from the intracellular Hsp90α, because
the plasma Hsp90α lacks four amino acid residues at its C-terminus
compared with the intracellular Hsp90α.

[0016] Therefore, in one aspect, this invention provides an isolated
polypeptide comprising or consisting of the amino acid sequence of SEQ ID
No.1.

[0017] The polypeptide of this invention may be phosphorylated. In
particular, in the polypeptide of this invention, one or more amino acid
residues in the amino acid sequence of SEQ ID No.1 selected from the
group consisting of Thr90, Ser231, Ser263, Tyr309 and a combination
thereof are phosphorylated. Preferably, the Thr90 in the polypeptide of
this invention is phosphorylated.

[0018] The polypeptide of this invention may serve as a tumor biomarker.
Using an agent specifically binding to the polypeptide of this invention,
it is possible to detect the plasma level of this polypeptide, and
thereby to determine the presence of cancers and the stage and metastasis
of cancers.

[0019] Accordingly, in another aspect, this invention relates to a method
of determining the presence, stage and/or metastasis of a cancer in a
subject, the method comprises the step of detecting the level of a
polypeptide comprising or consisting of the amino acid sequence of SEQ ID
No.1 in a plasma sample of the subject by using an agent capable of
specifically binding to said polypeptide.

[0020] In yet another aspect, this invention relates to a method of
screening the presence of a cancer in a high-risk population comprising
the step of detecting the level of a polypeptide comprising or consisting
of the amino acid sequence of SEQ ID No.1 in a plasma sample by using an
agent capable of specifically binding to said polypeptide.

[0021] In still another aspect, this invention relates to a method of
determining the prognosis of a patient having a cancer comprising the
step of detecting the level of a polypeptide comprising or consisting of
the amino acid sequence of SEQ ID No.1 in a plasma sample of the patient
by using an agent capable of specifically binding to said polypeptide.

[0022] In a further aspect, this invention relates to method of
determining the efficiency of a surgery, radiotherapy or chemotherapy
treatment to a patient having a tumor comprising the step of detecting
the level of a polypeptide comprising or consisting of the amino acid
sequence of SEQ ID No.1 in a plasma sample of the patient by using an
agent capable of specifically binding to said polypeptide.

[0023] Preferably, the agent capable of specifically binding to the
polypeptide of this invention can be a specific antibody against the
polypeptide. Preferably, the antibody is a monoclonal antibody or an
antigen binding fragment thereof, such as scFv, Fab, Fab' and F(ab')2. In
one specific embodiment, the antibody is MAb E9 or D10 produced by the
cell line deposited under CGMCC No. 2903 or 2904, respectively.

[0024] According to this invention, the antibody specifically binds to the
polypeptide present in plasma. Preferably, the antibody specifically
binds to a phosphorylated form of the polypeptide of the invention, said
phosphorylated form of the polypeptide contains one or more
phosphorylated amino acid residues in the amino acid sequence of SEQ ID
No.1 selected from the group consisting of Thr90, Ser231, Ser263, Tyr309
and a combination thereof. Preferably, the antibody specifically binds to
the polypeptide which is phosphorylated at Thr90.

[0025] In another aspect, this invention relates to a method of preventing
or treating cancer metastasis in a subject comprising the step of
administering an inhibitor of the polypeptide of the invention to the
subject. In one embodiment of this aspect, the inhibitor is a specific
antibody against a polypeptide comprising or consisting of the amino acid
sequence of SEQ ID No.1. Preferably, this antibody is humanized antibody
or an antigen binding fragment thereof. In one embodiment, the antibody
specifically binds to a phosphorylated form of the polypeptide, said
phosphorylated form of the polypeptide contains one or more
phosphorylated amino acid residues in the amino acid sequence of SEQ ID
No.1 selected from the group consisting of Thr90, Ser231, Ser263, Tyr309
and a combination thereof. In a preferred embodiment, the antibody
specifically binds to the polypeptide which is phosphorylated at Thr90.
In one specific embodiment, the antibody is MAb E9 or D10 produced by the
cell line deposited under CGMCC No. 2903 or 2904, respectively.

[0027] This invention also involves antibodies that can specifically bind
to the polypeptide of the invention which is present in plasma. In one
specific embodiment, the antibody is MAb E9 or D10 produced by the cell
line deposited under CGMCC No. 2903 or 2904, respectively. Preferably,
the antibody of the invention is a humanized antibody or an antigen
binding fragment thereof. In one embodiment, the antibody specifically
binds to a phosphorylated form of the polypeptide, said phosphorylated
form of the polypeptide contains one or more phosphorylated amino acid
residues in the amino acid sequence of SEQ ID No.1 selected from the
group consisting of Thr90, Ser231, Ser263, Tyr309 and a combination
thereof. In a preferred embodiment, the antibody specifically binds to
the polypeptide which is phosphorylated at Thr90.

[0028] In another aspect, this invention relates to a method of inhibiting
cancer invasiveness and metastasis, comprising the step of inhibiting the
phosphorylation of intracellular Hsp90α in cancer cells. In one
embodiment, this invention relates to a method of inhibiting cancer
invasiveness and metastasis, comprising the step of inhibiting the
phosphorylation of Hsp90α at Thr90 in cancer cells. In one specific
embodiment of this aspect, the method comprises a step of overexpressing
a nucleic acid molecule encoding protein phosphatase 5 in cancer cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1: The plasma Hsp90α level in tumor-bearing mice is
significantly increased than in that in normal mice.

[0030] FIG. 2: The plasma Hsp90α level in cancer patients is
significantly increased compared with that in normal people.

[0031]FIG. 3: The titer assay of the mouse monoclonal antibody of E9 and
D10.

[0034] A: The plasma Hsp90α level of liver cancer patients is
measured by sandwich ELISA. The plasma Hsp90α level in benign tumor
patients ranges from 2-10 ng/ml, mostly 2-5 ng/ml, while the plasma
Hsp90α level in 69% (20/29) liver cancer patients is above 50
ng/ml, the average of which has more than 10-fold increment compared with
that of benign tumor patients, which is consistent with the result of
Western blotting. This indicates that the plasma Hsp90α level is
positively correlated with tumor malignancy.

[0035] B: The plasma Hsp90α level of lung cancer patients is
measured by sandwich ELISA. The plasma Hsp90α level in 64% (9/14)
lung cancer patients is above 50 ng/ml, the average of which has more
than 10-fold increment compared with that of benign tumor patients. This
indicates the plasma Hsp90α level is positively correlated with
tumor malignancy.

[0036] C: The plasma Hsp90α level of breast cancer patients is
measured by sandwich ELISA. Compared with benign tumor patients, the
highest plasma Hsp90α level is increased by more than 5-fold.
Overall, the average plasma Hsp90α level in breast cancer patients
shows significantly increment when compared with that in benign tumor
patients.

[0037] D: The plasma Hsp90α level of pancreatic cancer patients is
measured by sandwich ELISA. The plasma Hsp90α level in 100% (10/10)
pancreatic cancer patients is above 50 ng/ml, the average of which has
more than 10-fold increment compared with that of benign tumor patients.
This indicates the plasma Hsp90α level is positively correlated
with tumor malignancy.

[0038] FIG. 6: Quantitative measurement of plasma Hsp90α level in
the cancer patients with or without metastasis using sandwich ELISA:

[0039] A: The liver cancer patients are divided into two groups, one with
metastasis and the other one without metastasis, then the plasma
Hsp90α levels in these two groups are compared. The plasma
Hsp90α levels in cancer patients with metastasis are all above 200
ng/ml while those in patients without metastasis range from 50-200 ng/ml.

[0040] B: The lung cancer patients are divided into two groups, one with
metastasis and the other one without metastasis, then the plasma
Hsp90α levels in these two groups are compared. The plasma
Hsp90α levels in cancer patients with metastasis are all above 200
ng/ml while those in patients without metastasis range from 50-200 ng/ml.

[0041] C: The breast cancer patients are divided into two groups, one with
metastasis and the other one without metastasis, then the plasma
Hsp90α levels in these two groups are compared. The plasma
Hsp90α levels in cancer patients with metastasis are significantly
elevated compared with that in patients without metastasis.

[0042] FIG. 7: Quantitative measurement of plasma Hsp90α level in
the patients with inflammation (pneumonia and hepatitis), normal people
and tumor patients using sandwich ELISA:

[0043] A: To ensure that the elevated plasma Hsp90α level of cancer
patients is tumor specific, we compared the plasma Hsp90α level in
pneumonia patients, normal people and tumor patients and found that the
plasma Hsp90α level in pneumonia patients ranges from 2-10 ng/ml,
indicating no significant changes compared with that in normal people.

[0044] B: The plasma Hsp90α level in hepatitis patients (Hepatitis A
and B) ranges from 2-10 ng/ml, indicating no significant changes compared
with that in normal people.

[0045] FIG. 8: The plasma Hsp90α is secreted by tumor cells.

[0046] FIG. 9: The secreted Hsp90α by tumor cells is in a C-terminal
truncated form.

[0050] FIG. 13: In the plasma of tumor patient, the increment of Thr90
phosphorylated Hsp90α level is consistent with that of Hsp90α
level.

[0051] FIG. 14: Thr90 Phosphorylation of Hsp90α is a pre-requisite
for Hsp90α secretion.

[0052] FIG. 15: PP5 dephosphorylates Thr90 phosphorylated Hsp90α.

[0053] A: Purified PP5 and Thr90 phosphorylated Hsp90α
(pT90-Hsp90α) are incubated together, and the released free
phosphate group is quantitatified. Peptide is a positive control. The
result shows PP5 can directly dephosphorylate Thr90 phosphorylated
Hsp90α.

[0054] B: In the human breast cancer cell line MCF-7, overexpression of
human PP5 results in the inhibition of Hsp90αThr90 phosphorylation
(0.55 of that in control group) and siRNA of endogenous human PP5 results
in the increase of Hsp90αThr90 phosphorylation (1.58 of that in
control group)

[0055] FIG. 16: PP5 regulates the secretion of Hsp90α.

[0056] A: In the human breast cancer cell line MCF-7, overexpression of
human PP5 results in the inhibition of Hsp90α secretion.

[0057] B: In the human breast cancer cell line MCF-7, siRNA of endogenous
human PP5 results in the increase of Hsp90α secretion.

[0058] FIG. 17: The correlation of PP5 expression level and the secretion
level of Hsp90α.

[0059] FIG. 18: The correlation of PP5 expression level and the invasive
ability of the tumor cells.

[0060] FIG. 19: The specific antibody of Hsp90α could inhibit the
migration of tumor cells.

[0064] Carcinogenesis is caused by changes of certain intracellular signal
transduction pathways, accompanied by changes of protein expression,
modification and distribution. These changes could be used to monitoring
of tumor development and progression, and these proteins are named as
tumor marker. With the development of proteomics technology, it becomes
possible to monitor the changes of tumor proteome qualitatively or
quantitatively. So many new tumor markers have been found to provide more
accurate and credible evidences for the tumor clinical diagnosis and
prognosis.

[0065] This invention is based on a discovery of a novel blood tumor
marker, i.e., plasma Hsp90α. Compared to intracellular Hsp90α
(the amino acid sequence is SEQ ID No.3, coding nucleic acid sequence is
SEQ ID No.4), the Hsp90α in plasma has a deletion of 4 amino acids
in the C terminus.

[0066] Hence, in one aspect, this invention provides an isolated
polypeptide, which is Hsp90α found in serum or plasma. In this
application, the term "polypeptide of the invention" used herein refers
to Hsp90α found in serum or plasma, which comprises or consists of
the amino acid sequence of SEQ ID No.1. Preferably, the term "polypeptide
of the invention" used herein refers to the polypeptide consisting of the
amino acid sequence of SEQ ID No.1 sequence. In this application, the
term "Hsp90α in plasma" or "Hsp90α in serum" can be used
equally to refer to the protein Hsp90α in the blood but not
intracellular or on cell surface. In this application, term "polypeptide"
and "protein" can be used interchangeably.

[0067] The present invention also provides a polynucleotide encoding a
polypeptide composing or consisting of the amino acid sequence of SEQ ID
No.1. In a specific embodiment, the polynucleotide comprises or consists
of the amino acid sequence of SEQ ID No.2.

[0068] The inventors also found that the polypeptide of the invention is
in a phosphorylated form in plasma. Specifically, one or more amino acid
residues in the amino acid sequence of SEQ ID No.1 selected from the
group consisting of Thr90, Ser231, Ser263, Tyr309 and a combination
thereof are phosphorylated. Preferably, the Thr90 in the polypeptide of
this invention is phosphorylated.

[0069] The special form of plasma Hsp90α(C-terminal truncated and
phosphorylated) has not been described. Meanwhile, plasma Hsp90α
also has never been reported to be related to tumor development and
progression. EP1457499A1 describes the extracellular form of
Hsp90α, and suggests that the inhibitors of Hsp90α can be
used to treat tumor metastasis, to detect tumor invasiveness and to
determine the dependence of tumor invasiveness on Hsp90α. However,
the method described in EP1457499A1 is used to detect cell surface
Hsp90α, but not related to plasma Hsp90α. Nor did EP1457499A1
disclose that plasma Hsp90α can be used to determine the degree and
stage of malignancy, or to monitor the therapeutic response and prognosis
of cancer by measuring the level of Hsp90α in plasma.
WO/2008/070472 reported that the effects of the anti-cancer treatment
targeting Hsp90α could be determined through detecting Hsp90α
and its related factors in plasma, but it did not mention Hsp90α as
an independent tumor marker in cancer diagnosis and prognosis.

[0070] The inventors examined the blood samples from nearly a hundred
tumor patients (with breast cancer, liver cancer, pancreatic cancer, lung
cancer and so on), and found that the level of Hsp90α in plasma is
correlated to tumor malignancy, particularly metastasis; while the
inflammatory response does not influence plasma level of Hsp90α.
Therefore, plasma Hsp90α can serve as a tumor marker, which can be
used for the diagnosis and prognosis of tumors and metastasis.

[0071] Additonally, the invention provides a kit examining the plasma
levels of the polypeptide of the invention. The kit of the invention
contains an agent capable of specifically binding to the polypeptide of
the invention, which can be used to detect the level of plasma
Hsp90α.

[0072] The invention also provides a method of detecting the level of
plasma Hsp90α in a plasma sample of the subject by using an agent
capable of specifically binding to said polypeptide. The method of the
invention can be used to establish the diagnosis of carcinogenesis,
malignancy and metastasis; to screening cancer in high-risk population;
to determine the prognosis of cancer patients; and to determine the
efficacy of surgery, radiotherapy or drug therapy.

[0073] As used herein, the term "an agent capable of specifically binding
to a polypeptide of the invention" refers to molecules which can bind to
the polypeptide of this invention with high-affinity. These agents also
include molecules which can bind intracellular and cell surface
Hsp90α. An agent capable of specifically binding to the polypeptide
of the invention can be proteins, especially Hsp90α specific
antibodies. In the preferred examples, the above antibody is a monoclonal
antibody or antigen binding fragment, such as scFv, Fab, Fab `and F
(ab`)2. In a specific embodiment, the antibodies are monoclonal antibody
E9 or D10 which is produced by the cell line with deposition number of
CGMCC No. 2903 or 2904, respectively.

[0074] Monoclonal antibodies are obtained by screening the cells which can
secret the antibodies and then culturing the cells in vitro. The method
is well known by the people in this field (Kohler G & Milstein C. (1975)
Nature. 256, 495-7). The process for Hsp90α-specific monoclonal
antibody preparation is as follows: for the first immunization,
recombinant human Hsp90α (rhHsp90α, 100 μg) with Freund's
complete adjuvant was injected subcutaneously on the back of BALB/c mice
multi-pointly; 3 weeks later, same dose of rhHsp90α was injected
intraperitoneal (i.p.) with Freund's incomplete adjuvant; the third
immunization was administered by i.p. injection after 3 weeks with same
dose (5 to 7 days later the blood titer was tested); the other 3 weeks
later, 200 μg rhHsp90α was administered for the booster
immunization by intraperitoneal injection. 3 days later, spleen cells
were fused with hybridoma SP2/0-Ag14 (SP2/0) (Source: ATCC, number:
CRL-1581) using HAT for screening. Then hybridoma cells were limited
diluted. immune blot and ELISA methods were used to identify and
eventually to select cell lines that can secrete specific Hsp90α
antibodies.

[0075] According to the invention, the antibodies used for the preparation
of the method or kit can specifically bind to Hsp90α, and
preferably to Hsp90α in plasma. In an preferred embodiment, the
antibodies specifically bind to Hsp90α with one or more following
amino acid residues are phosphorylated: Thr90, Ser231, Ser263, Tyr309 and
the combination thereof. In the preferred embodiment, antibodies
described herein can specifically bind to Hsp90α with
phosphorylated Thr90.

[0076] The invention also provides a method for detecting the level of
plasma polypeptide. The level of plasma Hsp90α can be detected by
any suitable methods. The methods described here include both direct and
indirect means for detecting the polypeptide which can be used for the
diagnosis of tumor development, malignancy and metastasis.

[0077] Direct measurements include methods of detecting the described
polypeptide of the invention using its an agent capable of specifically
binding to said polypeptide, for example using specific antibodies of the
polypeptides by Western blot or ELISA.

[0078] The concentration of Hsp90α can also be indirectly determined
by detecting the activity of Hsp90α. An example is the assay of
thermal induced denaturation of luciferase, which can be used to detect
the chaperone activity of Hsp90α (Johnson et al. (2000) J. Biol.
Chem., 275, 32499-32507).

[0079] Preferably, the level of plasma Hsp90α can be detected by
ELISA or Western blot, comprising the steps as follows: [0080] a)
collecting the whole blood of cancer patients, and obtaining plasma or
serum by centrifugation; [0081] b) using ELISA or Western blot to detect
the Hsp90α level of plasma or serum obtained from the step a), in
which plasma of healthy people is used as the negative control, while
plasma of patients with malignant tumors is used as the positive control,
optionally generating an Hsp90α concentration standard curve;
[0082] c) determining the tumor malignancy and stage according to the
level of plasma Hsp90α, thereby determining tumor diagnosis,
prognosis or efficacy of treatment.

[0083] In step b) other methods can also be used, such as methods based on
antigen-antibody reactions as well as methods based on other principles
directly or indirectly reflecting the concentrations of Hsp90α, for
example the examination of the concentration of Hsp90α by detecting
the activity of Hsp90α.

[0084] Standard Hsp90α used for the standard curve of ELISA is
purified from the plasma of cancer patients and can also be obtained by
recombinant construction, including the full length Hsp90α,
fragments, and other recombinant proteins or conjugates containing the
sequence of the Hsp90α. "the standard curve of Hsp90α
concentration" refers to the correlation curve between Hsp90α
concentration and absorbance values detected in the ELISA using standard
Hsp90α samples. "Hsp90α standard" refers to the plasma
Hsp90α protein, recombinant Hsp90α protein, fragments and
derivatives with the purity of more than 95%.

[0085] "Determining the malignancy of tumor" refers to making a judgment
of tumor malignancy by examining the Hsp90α concentration in the
plasma samples of patients and comparing this value with negative and
positive controls.

[0086] Both sandwich and competitive ELISA can be used to detect the level
of Hsp90α in plasma. The competitive ELISA with higher sensitivity
is preferred.

[0087] The general steps of Sandwich ELISA include: a) immobilizing a
specific antibody onto a solid support, and removing the unbound
antibodies and impurities by one or more washing steps; b) adding samples
to be tested and incubating for a period of time, allowing the antigen in
the samples to bind to the antibody immobilized on the solid support and
form solid-phase antigen-antibody immune complexes, then removing the
unbound substances; c) adding an enzyme-conjugated-antibody which can
bind to the antigen in the solid phase immune complexes, and then
removing the unbound enzyme-conjugated-antibody by thorough washes (the
amounts of enzyme-conjugated-antibody bound to the solid support are
positively correlated to the amount of antigen); and d) adding substrate
and quantifying the amount of antigen according to the color reaction.

[0088] The general steps of competitive ELISA include: a) immobilizing a
specific antibody onto a solid support and washing thoroughly; b) adding
the mixture of the sample to be tested and a certain amount of
enzyme-conjugated-antigen, and incubating for enough time and wash
thoroughly. (If there is no antigen in the sample, the
enzyme-conjugated-antigen can bind to the solid phase antibody without
competition. If there are antigens in the sample, then the antigens in
the sample compete with the enzyme-labeled-antigens to bind the
immobilized antibody, and the amount of enzyme-labeled-antigen bound to
immobilized antibody decreases. As a control, the reference tubes only
contain the enzyme-labeled antigen); c) adding substrate and obtaining
the absorbance value for each tube. The value of the reference tube is
highest, and the difference between the reference tube and the sample
tube represents the amount of antigens in the sample. The lighter the
color is, the more antigens the sample contains.

[0089] If Sandwich ELISA is used, the antibodies are two different species
of plasma Hsp90α specific antibodies. The immobilized antibody can
be a rabbit polyclonal antibody with strong binding capacities, while the
detection antibody is a mouse monoclonal antibody with high specificity.
These two antibodies should have no cross-reaction.

[0090] If competitive ELISA is used, the antibodies should be the specific
antibodies against plasma Hsp90α, which should have strong binding
capacities and high specificity to both the competitve substance and
Hsp90α.

[0091] If competitive ELISA is used, the competitive substances can be a
labled plasma Hsp90α standard protein. And the labeling does not
interfere with the binding of Hsp90α standard protein to its
antibody.

[0092] If Western blot method is used, the first antibody used is a
specific antibody against plasma Hsp90α; the secondary antibody can
be conjugated with horseradish peroxidase, or alkaline phosphotase; the
substrate can be DAB or fluorescent substrate. The fluorescence substrate
with high sensitivity is preferred.

[0093] The sensitivity of above methods should be 10 ng/ml or more
sensitive.

[0094] The antibody specific for plasma Hsp90α used in this present
invention can be a whole antibody, or its fragments and derivatives.

[0095] The antibodies of this invention can also be replaced by other
agents capable of specifically binding to Hsp90α, wherein the
agents include small molecule compounds, peptides and their derivatives.

[0096] The competitive plasma Hsp90α standard proteins can be
labeled by biotin and various fluorescent labeling reagents. Biotin is
preferred.

[0098] The inventors demonstrated that, the plasma Hsp90α level in
non-cancer human is 2-50 ng/ml, most in the range of 2-10 ng/ml. The
level of plasma Hsp90α in patients diagnosed with cancer is higher
than the normal level, while the plasma Hsp90α level in the
patients having cancer metastasis is higher than 50 ng/ml, mostly higher
than 200 ng/ml. This makes the plasma Hsp90α a new tumor marker,
which would be helpful for the diagnosis of cancer, especially
metastasis.

[0099] Thus, in an embodiment, the kit or method of this invention can be
used to determine the existence of tumor, especially malignant tumors and
tumor metastasis. To this end, the kit or method of this invention can be
used to measure the Hsp90α level of plasma samples from potential
tumor patients or potential tumor metastasis patients, and optionally
compared with normal controls, and then determine the possibility of
patients with tumor or tumor metastasis according to the Hsp90α
level in the sample. The elevated Hsp90α level in plasma suggests
higher possibility that the patients have suffered from malignant tumor,
and for patients with known cancer, an elevated Hsp90α level
strongly suggests possibility of tumor metastasis.

[0100] In another embodiment, the kit or method of the invention can be
used to detect the level of plasma Hsp90α so as to screen high-risk
populations. To this end, the kit or method of the invention can be used
to detect plasma Hsp90α level in samples from high-risk
populations, and optionally compared with normal controls, and then one
can determine whether an individual within the population may have tumor
according to the level of the sample. Elevated Hsp90α level
indicates the higher possibility of the presence of a malignant tumor. It
is well-known for the people in this field how to determine different
high-risk groups depending on the type of cancer to be screened,
individual factors such as age, family history, lifestyle, work
environment, history of exposure to harmful compounds and so on. For
example, patients having chronic hepatitis B or hepatitis C are at high
risk of HCC.

[0101] In another embodiment, the kit or method of the invention can be
used to detect the level of plasma Hsp90α to predict the prognosis
of cancer patients. To this end, the kit or method of this invention can
be used to detect plasma Hsp90α level in samples from cancer
patients, and optionally compared with normal controls, and then one can
determine the prognosis of cancer patients according to the level of
Hsp90α in plasma samples. The maintenance of Hsp90α in a high
level or further increase may relate to the poor prognosis. Therefore,
the clinicians can be alerted to provide more closely observation to the
patients, and if necessary, change the current treatment.

[0102] In another embodiment, the kit or method of the invention can be
used to detect the plasma level of Hsp90α, which is thus used to
determine whether the treatment such as the surgery, radiotherapy or
chemotherapy on cancer patients is effective. To this end, the kit or
method of this invention can be used to detect plasma Hsp90α level
in samples from cancer patients, and optionally compared with normal
controls, and then one can determine the prognosis of cancer patients
according to the level of Hsp90α in plasma samples. According to
the level of the Hsp90α, one can determine whether the surgery,
radiotherapy or chemotherapy on cancer patients is effective and/or
whether the treatment should be continued.

[0103] The inventors further demonstrated that the Hsp90α in plasma
is secreted by tumor cells and is different from that within the tumor
cells. Therefore, it is assumed that the tumor development and metastasis
may be suppressed by inhibiting the secretion of Hsp90α into
plasma. This point was demonstrated by the mouse tumor metastasis model
using the specific antibody against plasma Hsp90α. Therefore, the
secretion of Hsp90α can be used as a new target for screening new
anticancer drugs.

[0104] In another aspect, the invention provides a method for the
preventing or treating tumor metastasis comprising administering to a
cancer patient an inhibitor of the polypeptide of the invention.

[0105] According to one embodiment of this invention, the aforementioned
inhibitor is a specific antibody of the polypeptide. Preferably, the
antibody is a humanized antibody or its fragments. In one embodiment,
these antibodies specifically bind to a phosphorylated form of the
polypeptide of the invention, said phosphorylated form of the polypeptide
contains one or more phosphorylated amino acid residues in the amino acid
sequence of SEQ ID No.1 selected from the group consisting of Thr90,
Ser231, Ser263, Tyr309 and a combination thereof. In a preferred
embodiment, the antibody can bind to the polypeptide phosphorylated at
Thr90. In one specific embodiment, the antibody is MAb E9 or D10 which is
produced by the cell line deposited under CGMCC No. 2903 or 2904,
respectively. As demonstrated herein, these antibodies can completely
inhibit the lymph node metastasis of tumors in mouse model and can
inhibit the lung metastasis to an extent up to 56%.

[0106] So, in another aspect, this invention also relates to an antibody
specifically binds to the Hsp90α in blood. In a specific
embodiment, the antibody is MAb E9 or D10 which is produced by the cell
line deposited under CGMCC No. 2903 or 2904, respectively. Preferably,
the antibody is a humanized antibody or an antigen binding fragment
thereof. In a specific embodiment, the antibody specifically binds to a
phosphorylated form of the polypeptide of the invention, said
phosphorylated form of the polypeptide contains one or more
phosphorylated amino acid residues in the amino acid sequence of SEQ ID
No.1 selected from the group consisting of Thr90, Ser231, Ser263, Tyr309
and a combination thereof. In one preferred embodiment, the antibody
specifically binds to the polypeptide phosphorylated at Thr90.
Preferably, the antibody inhibits tumor growth, especially metastasis.
This invention also related to a conjugate comprising the antibody and a
diagnosis or treatment moiety. For example, the diagnosis moiety is a
fluorophore, while the treatment moiety is a chemotherapeutic agent.

[0107] The inventor also found the amount of Hsp90α secreted by
tumor cells is related to the level of Protein phosphatase 5 (PP5). In
benign tumor, secreted Hsp90α is low but PP5 expression level is
high. In malignant tumor, secreted Hsp90α is high but PP5
expression level is low. Therefore, the level of secreted Hsp90α is
negatively interrelated with the level of PP5. As a result, by detecting
the expression level of PP5, it is possible to predict the amount of
secreted Hsp90α.

[0108] The inventors also found that the secretion of Hsp90α can be
inhibited by cellular PP5. Over-expression of a nucleic acid molecule
encoding PP5 in the cell can inhibit Hsp90α secretion, while a
decrease in the expression level of PP5 will result in an increase in
Hsp90α secretion. So by over expressing PP5, Hsp90α secretion
can be inhibited and the tumor progression and metastasis may be
inhibited. According to our experiment, we found that over expression of
PP5 inhibits the metastasis of MCF-7. So PP5 can be a new therapeutic
target for tumor treatment.

[0109] So, in a further aspect, this invention provides a method for
inhibiting tumor invasiveness and metastasis, wherein a step of
inhibiting the phosphorylation of Hsp90α within tumor cells. In one
embodiment, this method comprises a step of inhibiting Thr90
phosphorylation of Hsp90α in tumor cells. In one specific
embodiment, this method comprises a step of over-expressing a nucleic
acid molecule encoding PP5 in tumor cells. Preferably, the
over-expression of PP5 is achieved by the means of gene introduction. In
one embodiment, this method comprises a step of over-expressing a nucleic
acid molecule encoding PP5 having an amino acid sequence of SEQ ID No.5.
In one specific embodiment, the nucleic acid molecule comprises the
nucleotide sequence of SEQ ID No.6. This invention also provides a method
of inhibiting the phosphorylation of Hsp90α in tumor cells
comprising a step of over-expressing PP5 in tumor cells using a vector
carrying a polynucleotide encoding PP5 operably linked to a promoter. The
method can be used to inhibit tumor invasiveness and metastasis.

[0110] This invention also provides methods and models for screening for
anti-tumor drugs by using plasma Hsp90α and its derivatives. Such
anti-tumor drugs includes, but not limited to, plasma Hsp90α
binding proteins, small peptides, and small compounds aswell as
inhibitors which can suppress the activity of plasma Hsp90α.

EXAMPLES

Example 1

The Collection and Preparation of Mouse Plasma Samples, and the Detection
of Plasma Hsp90α

[0111] Balb/c mice with average body weight of 20 gram (purchased from
Beijing Vital River Laboratory Animal Technology Co., Ltd.) were
randomized divided into two groups, 3 for each. The mice of experiment
group were inoculated with 106 H22 (CCTCC ID: GDC091) cells, while
the mice in control group were not inoculated. When the diameter of tumor
reached about 2 cm (about 20 days), blood was collected from eye down
veniplex. Anticoagulant was added and hemolysis was prevented. If
hemolysis occurred, the blood would be recollected. The blood sample was
centrifuged at 4° C. with 6000 g for twice, and supernatant was
saved. The amount of plasma Hsp90α was detected by Western blotting
using Rabbit anti-human Hsp90α pAb (Labvasion). BCA method was used
to determine the total amount of proteins in samples, which ensured that
the loading amounts of different samples were equal. The result is shown
in FIG. 1. Compared to control mice, the plasma Hsp90α was elevated
in tumor bearing mice.

Example 2

The Collection and Preparation of Plasma Samples from Normal People and
Tumor Patients, and the Detection of Plasma Hsp90α

[0112] The blood of normal people and cancer patients were collected and
delivered to lab within 24 hours at 4° C. If hemolysis occurred,
the samples should be recollected. The samples were centrifuged at
4° C., 6000 g twice and supernatant was saved. The amount of
Hsp90α was determined by Western blotting. If the samples could not
be examined immediately, the samples should be stored at -80° C.
By comparing the results with clinical diagnosis, the correlation between
Hsp90α and tumor malignancy was confirmed.

[0113] The protocol for Western blotting: the plasma samples were mixed
1:1 with loading buffer. 1-2 μl sample is loaded for SDS-PAGE. Primary
antibody is the one which can specifically recognize plasma
Hsp90α(rat mAb SPA-840, Stressgen). The aecondary antibody is goat
anti-rat antibody conjugated with HRP (purchased from Zhongshanjinqiao).
As the result shows in FIG. 2, the amount of Hsp90α in HCC patients
is 10-fold higher than that of normal people (A), while it is elevated by
2-fold in benign galactocele and hysteromyoma patients compared with that
of normal people.

Example 3

Preparation of Rabbit pAb and Mouse mAb against Hsp90α

[0114] Primers with the following seauences were used to clone the gene of
Hsn90α:

Primers were synthesized by Invitrogen. Pfu DNA polymerases (NEB) were
used to amplify Hsp90α from human liver cDNA library (Stratagene).
Sph1 and Sal1 (NEB) were used to digest the amplified PCR products and
pQE80L vector (Qiagen). Then T4 ligases were used for ligation. The
products were transformed into Top10 cells (Transgen) for amplification
and validation. The validated plasmids were further transformed into
BL21DE3 cells (Transgen) for expression. The recombinant Hsp90α
proteins were purified by ion-exchange chromatography SP HP, pH6.8,
collecting 10 ms/ml peak and Q HP, pH7.8, collecting 19 ms/ml peak.

[0115] Recombinant human Hsp90α with a purity higher than 95% was
used to immune adult male New Zealand Rabbit by dorsal subcutaneous
multi-point injection at 100 μg for each time. Two weeks later, the
secondary immunization was conducted according to the same method, except
that the amount of Hsp90α injection was reduced to 50 μg. Boost
injections were conducted every 1 week after the secondary immunization
for twice. The titer of antibody in serum was determined 7-10 days after
the boost immunizations. Eight days after the last immunization, serum
was collected and stored at -20° C. Affinity chromatography
conjugated with antigen was used to purify the antibody from the serum.
Purified rabbit pAb was named as S2.

[0116] BALB/C mice were immunized by recombinant human Hsp90α.
Primary immunity: 100 μg antigen and Freund's complete adjuvant was
injected dorsal subcutaneously multipoint. The second immunity was
conducted 3 weeks later with Freund's incomplete adjuvant using the same
dose and i.p. injection. The third immunization was operated 3 more weeks
later without adjuvant (blood was collected for test after 5-7 days).
Three more weeks later, 200 μg antigen was injected i.p. as the boost
immunization. 3 days later, spleen cells were collected and fused with
SP2/0-Ag14(SP2/0) hybridomas (ATCC: CRL-1581). HAT was used for the
screening. Limited dilution was used to colonize hybridomas. Western
blotting and ELISA were used for identification. Finally, E9 and D10,
which secrete specific antibody against Hsp90α, were obtained and
stored as CGMCC No. 2903 and 2904 on Feb. 24, 2009.

[0117] Indirect ELISA was used to determine the titers of E9 and D10.
Shown as FIG. 3, the average titers of E9 and D10 respectively reaches
500,000, which is qualified to be used in the detection of plasma
Hsp90α. Indirect ELISA: recombinant human Hsp90α was plated
at 4° C. overnight with the concentration of 10 μg/ml. Then the
plate was blocked at 37° C. for 1 hour. E9 or D10 with 1:400,
1:1600, 1:6400, 1:25600, 1:102400, 1:509600 dilutions was added and
incubated for 2 hours at RT. Then goat anti-mouse antibody conjugated
with HRP was added and incubated for 1 hour at room temperature, then
o-phenylendiamine was added and absorbance at OD 490 nm was detected.

Example 4

Measurement of the Concentration of Hsp90α by E9, S2 and Sandwich
ELISA

[0118] In the method of sandwich ELISA to test the concentration of plasma
Hsp90α, two antibodies from distinct species were used. S2 with
high binding capacity (the preparation of S2 was described in example 3)
was used as the coating antibody and E9 (the preparation of E9 was
described in example 3) with high binding specificity was used as the
detecting antibody. There is no cross reaction between these two
antibodies. The method is repeatable and sensitive. FIG. 4 shows that the
sensitivity of this method is 5 ng/ml.

Example 5

The collection and Preparation of Human Plasma, the Detection of Plasma
Hsp90α and Determination of Tumor Malignancy (Sandwich ELISA)

[0119] The blood of normal persons, cancer patients and inflammation
patients was delivered to lab at low temperature within 24 hours. If
hemolysis occurs, the blood should be re-collected. The samples were
centrifuged at 4° C., 6000 g twice and supernatant was saved. The
amount of plasma Hsp90α was determined by ELISA. The samples were
stored at -80° C. if they were not examined immediately. By
comparing the results with clinical diagnosis, the correlation between
Hsp90α and tumor malignancy was confirmed.

[0120] Two different Hsp90α antibodies were used in sandwich ELISA.
S2 was coated on the plate and incubated overnight at 4° C. The
plate was blocked for 1 hour at 37° C. The samples were diluted by
10 folds and were added to the plate (100 μl/well). After 2 hours
incubation at 37° C., E9 was added and incubated at 37° C.
for 2 more hours. Goat anti-mouse antibody conjugated with HRP was added
to incubate for another 1 hour. O-phenylendiamine was added and
absorbance at OD490 nm was detected. The results were shown as FIGS. 5, 6
and 7. The standard curve comprises a serial of samples containing the
gradient concentration of standard Hsp90α proteins and 10% of
negative plasma in each sample, which was used to exclude the background
of plasma.

[0124] The normal people's samples were collected from healthy volunteers,
while tumor patients and inflammation patients' samples were collected
from Beijing Cancer Hospital and Xiamen First Hospital.

Example 6

The Plasma Hsp90α was Secreted by Tumor Cells

[0125] Nude mice (purchased from Beijing Vital River Laboratory Animal
Technology Co., Ltd.) with average body weight of 20 g were divided into
two groups, 6 mice for each group. Each mouse was injected with 106
Hela cells (ATCC: CCL-2). Control group were normal mice without tumor.
When the diameter of tumor reached 2 cm (about 20 days), blood was
collected from eye veniplex. Hsp90α antibody (rat mAb, Stressgen)
that specifically recognizes human Hsp90α but not mouse
Hsp90α was used to detect the plasma Hsp90α. As shown in FIG.
8, the plasma Hsp90α in the tumor-bearing mice is specifically
recognized by human but not mouse Hsp90α antibody, which indicates
that the plasma Hsp90α was secreted by the xenograft tumor cells.

Example 7

The Hsp90α Secreted by Rumor Cells was C-Terminally Truncated

[0126] Using Hsp90α-pc3.1-Nhe1-For-Myc: GCTAGCTAGCGCCACCATGGA
ACAAAAACTCATCTCAGAAGAGGATCTGCCTGAGGAAACCCAGACCCAAGAC (SEQ ID No.10) and
Hsp90α-pc3.1-Xho1-Re-nostop: CCCGCTCGA GTGTCTACTTCTTCCATGCGTGATG
(SEQ ID No.12) as primers (synthesized by Invitrogen) and Pfu DNA
polymerase (NEB) to amplify Hsp90α from the template of
pQE80L-Hsp90α (obtained in example 3). The products were digested
and inserted into pcDNA3.1/Myc-His(-) (Invitrogen) to obtain Hsp90α
with Myc tag at the N terminus, which was named as Myc-H. Similarly,
His-Myc-H with N-terminal tandem His-Myc tags was amplified by
Hsp90α-pc3.1-Nhe 1-For-His-Myc:
GCTAGCTAGCGCCACCATGCATCATCATCATCATCATGAACAAAAACTCATCTCAGA
AGAGGATCTGCCTGAGGAAACCCAGACCCAAGAC (SEQ ID No.11) and SEQ ID No.12
primers. These two plasmids were transiently transfected into MCF-7 to
observe the secretion of exogenous Hsp90α. Antibodies against
Hsp90α, Myc and His were used to detect the secreted Hsp90α.
The result shows that the secreted Hsp90α was C-terminally
truncated (FIG. 9A).

[0127] Using Myc-His-H as a template, the mutations of the final four
amino acids (EEVD) of Hsp90α C-terminus was constructed. EE->AA
represents the mutation of two EE to two Ala (using SEQ ID No.11 and
Hsp90α-EE-AA: GGCCGCTCGAGTGTCTACTGCTGCCATGCGTGATGTG (SEQ ID No.13)
as primers), VD->AA means that VD were mutated to two Ala (using SEQ
ID No.11 and Hsp90α-VD-AA: GGCCGCTCGA GTTGCTGCTTCTTCCATGCGTGATGTG
(SEQ ID No.14) as primers). All Ala represents the mutation of EEVD to
four Ala (using SEQ ID No.11 and Hsp90α-EEVD-AAAA:
GGCCGCTCGAGTTGCTGCTGCTGCCATGCG TGATGTG (SEQ ID No. 15) as primers), CM
represents the deletion of last EEVD four amino acids (using SEQ ID No.11
and Hsp90α-CΔ4-Xho: CCGCTCGAGTCATGCGTGATGTGTCGTCATCTC (SEQ ID
No.16) as primers). Human breast cancer cell line MCF-7 was transiently
transfected with these types of mutants to observe the secretion of
exogenous Hsp90α (over-expressed Hsp90α, which is different
from the endogenous one). The antibody against Hsp90α was used to
detect the changes of secreted Hsp90α in the extracellular medium.
The results show that four C-terminal amino acid residues regulate the
secretion of Hsp90α. Any site-directed mutation or deletion of
these four amino acid residues can lead to the secretion of Hsp90α
without C-terminal truncation, which suggests that four C-terminal amino
acid residues EEVD are deleted in the secreted extracellular Hsp90α
(FIG. 9B).

Example 8

Examination of the existing form of Hsp90α in human plasma

[0128] We collected whole blood samples of liver cancer patients, which
were centrifuged twice within 24 hours after collection, then detected
Hsp90α in plasma using the method of immunoprecipitation and
immunoblotting. First, specific rabbit polyclonal antibody against
Hsp90α (Source: Labvision) was used for immunoprecipitation, and
then a rabbit polyclonal antibody which can specifically recognize the
Hsp90α C-terminal four amino acid residues EEVD (lab stock, antigen
used for immunizationused is a carrier protein coupled with
three-repeated peptides of EEVD, synthesized from the SBS Genetech Co.,
Ltd.) was used to detect Hsp90α in plasma. As shown in FIG. 10, the
EEVD antibody specifically recognize Hsp90α from whole cell lysate,
but does not recognize Hsp90α in plasma, which indicates that
intracellular Hsp90α is different from plasma Hsp90α, which
lacks the four C-terminal amino acid residues EEVD. (FIG. 10).

Example 9

Detection of the Phosphorylated form of Hsp90α in Plasma

[0129] Whole blood samples of the liver cancer patients were centrifuged
twice to extract the plasma within 24 hours after collection. Then
Hsp90α in plasma was detected using the method of
immunoprecipitation and immunoblotting. First, specific rabbit polyclonal
antibody against Hsp90α (Source: Labvision) was used to
immunprecipitate plasma Hsp90α, then an antibody (Rabbit
anti-phospho-(Ser/Thr) PKA substrate pAb, Cell signaling) which can
specifically recognize the Thr90 phosphorylated Hsp90 was used to detect
the Thr90 phosphorylation status of plasma Hsp90. As shown in FIG. 11,
the Hsp90α in plasma is Thr90 phosphorylated.

Example 10

Detect the Concentration of Thr90 Phosphorylated Hsp90 in Plasma

[0130] Whole blood samples of both liver cancer patients and normal people
were centrifuged twice to extract the plasma within 24 hours after
collection. The relative level of Hsp90α in the plasma was detected
using the method of sandwich ELISA. Protocol: firstly, using self-made
rabbit polycolonal antibody S2 to coat the plate overnight at 4, then
added 10-fold diluted plasma samples at 100 μl per well. After
incubation at 37 for 2 hours, antibody which can specifically recognize
the Thr90 phosphorylated Hsp90α (cell signal) was added. The plate
is incubated at 37 for 2 hours; and then horseradish peroxidase
conjugated goat anti-rabbit secondary antibodies were added and incubated
for 1 hour. Finally o-phenylenediamine was added to detect the absorption
at OD490 nm. The results show that the levels of Hsp90α in liver
cancer patients are higher than those of normal people, P=0.003, Student
t test, which indicates that the level of Thr90 phosphorylated Hsp90 is
increased in liver cancer patients (FIG. 12).

Example 11

Detection of the Consistency of the Thr90 Phosphorylated Hsp90 Level in
Plasma and the Total Hsp90α Content

[0131] The levels of the Thr90 phosphorylated Hsp90 and the total amount
of Hsp90α in liver cancer patients plasma (8 cases) were detected.
The method to detect the total amount of plasma Hsp90α was the same
as that used in example 5; the method to detect the level of Thr90
phosphorylated Hsp90 was the same as that used in example 10. The results
show that the level of Thr90 phosphorylated plasma Hsp90 is consistent
with the total amount of plasma Hsp90α, which further indicates
that the phosphorylation of plasma Hsp90α is on Thr90, and the
increment of the total amount of Hsp90α can represent the increment
of Thr90 phosphorylated Hsp90α (FIG. 13).

Example 12

The Phosphorylation of Thr90 is Necessary for the Secretion of
Hsp90α

[0132] Using pcDNA3.1-Myc-His-Hsp90α plasmid as the template (Also
known as wild-type Hsp90α (WT Hsp90α)), mutant Hsp90α
(T90A, threonine mutated to alanine) was constructed by quickchange PCR
using the following primers:
Hsp90α-T89A-Sense:GATCGAACTCTTGCAATTGTGGATACTGGAATTGGAATG(SEQIDNo17-
) and Hsp90α-T89-AntiS nse: CATTCCAATTCCAGTATCCACAATTGCAAGAGT TCGATC
(SEQ ID No.18). T90A Hsp90α mutant can not be phosphorylated at
Thr90. Human breast cancer cell line MCF-7 (purchased from ATCC, No.
HTB-22) was transfected with wild-type Hsp90α (WT) or mutant
Hsp90α (T90A). The medium was then collected and the secretion of
exogenous Hsp90α was detected by anti-Hsp90α antibody.

[0133] The results show that overexpressed exogenous wild-type
Hsp90α can be detected in the extracellular medium, while the T90A
mutant can not be detected, which indicates that phosphorylation at
residue Thr90 is a prerequisite for Hsp90α secretion. (FIG. 14).

Example 13

PP5 is Responsible for the Dephosphorylation of pT90-Hsp90α

[0134] The preparation of the Thr90 phosphorylated Hsp90α
(pT90-Hsp90α): The recombinant human Hsp90α protein and
recombinant protein kinase A (Promega Corporation USA) were incubated in
a reaction buffer (NEB UK company) at 30° C. for 1 h, then
pT90-Hsp90α protein was purified. After removing free phosphate by
dialysis, the purified pT90-Hsp90α protein mixed with recombinant
human PP5 protein were incubated at 30° C., the free phosphate
released from the pT90-Hsp90α was detected using the
non-radioactive serine/threonine phosphatase assay kit (Promega
Corporation USA). Peptide substrate is a composition of the kit and was
used as the positive control. As shown in FIG. 15A, when PP5 was
incubated with the peptide substrate, the release of free phosphate was
significantly increased, P value<0.005, Student t test, indicating
that PP5 can directly dephosphorylate the peptide substrate (positive
control). However, when PP5 was incubated with pT90-Hsp90α protein,
the release of free phosphate was also significantly increased, P
value<0.005, Student t test, indicating that PP5 can directly
dephosphorylate pT90-Hsp90α.

[0135] The nucleotide sequence of PP5 (SEQ ID No.6) was amplified from
human liver cDNA library and constructed into the pcDNA3.1/Myc-His (-)
(vector source: Invitrogen). The PP5 vector was transfected into and
overecpressed in human breast cancer cell line MCF-7. On the other side,
using the RNA interference technology, the expression of PP5 can also be
knocked down using the siRNA with the sequence of
5'-ACTCGAACACCTCGCTAAAGAGCTC-3' (SEQ ID No.7) (synthesized by
Invitrogen). Then the status of Hsp90αThr90 phosphorylation was
examined with the overexpression or knock down of PP5. As shown in FIG.
15B, with overexpression of human PP5, the Thr90 phosphorylated
Hsp90α (pT90-Hsp90α) was significantly reduced (0.55 of the
control). When the expression of human PP5 was suppressed, the Thr90
phosphorylated Hsp90α (pT90-Hsp90α) was significantly
increased (1.58 of the control).

Example 14

Regulation of the Secretion of Hsp90α by Promoting or Inhibiting the
Expression of PP5

[0136] PP5 can dephosphorylate Thr90 phosphorylated Hsp90α. Primers
as follows were used to amplify PP5 from human liver cDNA library:
PP5-NheI-For: CTAGCTAGCATGTACCCATACGACGTCCCAGACTACGCT (SEQ ID No.19) and
PP5-XhoI-Re: CCGCTCGAGTTAATGATGATGATGATG ATGCACGTGTACC (SEQ ID No.20).
The full length human PP5 was cloned into pcDNA3.1/Myc-His (-) (vector
source: Invitrogen). Human breast cancer cell line MCF-7 was transfected
with PP5 vector, then the secretion of Hsp90α from the cells was
examined. The results showed that after overexpression of human PP5, the
secretion of Hsp90α was significantly decreased (FIG. 16A).

[0137] On the other side, when the expression of human PP5 in MCF-7 cells
was knocked down by RNA interference (against human PP5, Invitrogen), the
secretion of Hsp90α was significantly increase (FIG. 16B).

Example 15

The Level of PP5 and the Tumor Malignancy

[0138] The relationship between the expression level of PP5 and the
secretion of Hsp90α was examined in human breast cancer cell lines
MCF-7, SKBR3, MDA-MB-453, 435s and 231 (ATCC, number, respectively
HTB-22, -30, -131, -129, and HTB-26) using the method of Western
blotting. MCF-7, SKBR3 breast cancer cell lines are less malignant cell
lines. In the nude mice tumor model, these two cell lines can form
primary tumors, but do not metastasize. MDA-MB-453, 435s and 231 are more
malignant, They can not only form primary tumors, but also metastasize to
distant organs in the nude mice tumor models. Thus MDA-MB-435s and 231
are often used to establish tumor metastasis models. In FIG. 17,
secretion levels of Hsp90α by these five breast cancer cell lines
correlate with their malignancy.

[0139] The results show that the cells, which express high level of PP5,
secrete low level of Hsp90α; whereas cells with low expression of
PP5 can secrete more Hsp90α (FIG. 17). Meanwhile, the results also
show that the level of secreted Hsp90α is positively correlated,
whereas the expression level of PP5 is negtively correlated with the
tumor malignancy (FIG. 17). The level of secreted Hsp90α and its
regulatory factors such as PP5 can be used to determine the tumor
malignancy.

Example 16

The Expression Level of PP5 is Linked to Tumor Invasiveness

[0140] The wound healing model was employed to examine the relationship
between the expression level of PP5 and tumor cell migration.

[0141] Human breast cancer cell line MCF-7 was transfected with human PP5
vector or PP5 siRNA. Then the cells were inoculated into 12-well plate.
When the cells grew to confluence, pippete tips were used to scrape cells
to form a "wound". The scraped cells were washed away, and the rest part
of cells were cultured in fresh DMEM medium (GIBCO) at 37 in an incubator
with 5% of CO2. The images of the "wound" at the time of 0 h, 12 h,
and 24 h were captured (FIG. 18A). The effect of PP5 expression on cell
migration was examined by analyzing the "wound" healing rate. The results
show that overexpression of PP5 inhibits MCF-7 cell migration, while PP5
siRNA can promote MCF-7 cell migration (FIG. 18B).

[0142] The wound healing model was used to detect the effect of plasma
Hsp90α specific antibody on tumor cell migration.

[0143] MCF-7 or MDA-MB-231 cells (ATCC, No. HTB-22, respectively, and
HTB-26) were inoculated into 12-well plate. When the cells grow to
confluence, pippete tips were used to scrape cells to form a "wound". The
scraped cells were washed away, and the rest part of cells were moved
into fresh DMEM medium (GIBCO). Meanwhile, E9 (20 μg/ml), the specific
mouse monoclonal antibody against Hsp90α, or control IgG (20
μg/ml) was added. Then the plate was incubated at 37, with 5%
CO2. Images of the "wound" after 0 h, 6 h, 12 h, 24 h, 48 h, and 72
h of incubation were captured (FIG. 18A). The effect of plasma Hsp90
specific antibodies on tumor cells migration was examined by monitoring
the "wound" healing rate. As shown in FIG. 19, the specific antibody
against plasma Hsp90α can significantly inhibit the migration of
both MDA-MB-231 (FIG. 19A) and MCF-7 (FIG. 19B) (Inhibitory
activity>40%).

[0144] Nude mice with an average body weight of 20 g were purchased from
Beijing Vital River Laboratory Animal Technology Co., Ltd. B16/F10 mouse
melanoma cells (ATCC, number: CRL-6475) (2×105) were injected
into the mice via tail vein. Next day the mice were randomly divided into
two groups (n=8): the control group (IgG was administered) and the
Hsp90α Ab (mouse monoclonal antibody E9) treated group. The
antibodies were administered once every other day with a dosage of 40
μg/mouse/time. Metastasis was detected 15 days after inoculation. As
shown in FIG. 20, the specific antibody against plasma Hsp90α can
completely inhibit the lymph node metastasis of B16/F10 cells (A), and
56% of the lung metastasis can be inhibited (B).